GSM8047295: Pparg2, High fat diet, ZT6 MiSeq_rep2; Mus musculus; OTHER
4C templates were prepared from 0.5-0.7 g of mouse  liver tissues homogenized in PBS 1X pH 7.4, filtered in 70 µm cell strainer and diluted to 30 ml with PBS. Cells were pelleted and crosslinked in 25 ml of DMEM with 1% of formaldehyde at room temperature under rotation for 15 minutes and immediately quenching the reaction with glycine at 0.125 M final concentration for 5 minutes at 4°C. Nuclear isolation was performed in NI buffer (10mM Tris HCl, 10mM NaCl, 0.2% NP-40) for 1 h at 4°C. For first restriction, selected six-cutter enzymes were EcoRI for Pparγ2 bait, and HindIII for Dbp, Pparα and Srebp1c baits. Ten million nuclei per sample were resuspended in 500 µl of 1X NEBbuffer 2.1 (New England BioLabs) containing 0.3% SDS and incubated 10 min at 60°C and 50 min at 37°C. Triton X100 was added at 1.8% v/v, incubated 1 h at 37°C, and 400U of restriction enzyme were added and incubated at 37°C overnight with agitation. First ligation was performed with 100U of T4 DNA ligase (ThermoScientific Cat. No. EL0011) with 5% polyethylenglicol in 7ml final volume, during 6h at 16°C. DNA crosslinking was reversed by adding 30 µl of 10 mg/ml proteinase K (EMD Millipore Cat. No. 124568) and incubating 6h at 65°C, followed by addition of 30 µl of 10 mg/ml RNAse A (Sigma Cat. No.R5000) and incubation 45 min at 37°C. DNA was purified with phenol/chloroform extraction and sodium acetate/ethanol precipitation. The purified DNA had a second restriction with 200U of Csp6I (New England BioLabs) in 500 µl reaction volume. Finally, a second round of ligation with 200U de T4 DNA ligase with 5% PEG in a 14 ml final volume was performed. Circular DNA was resuspended in molecular grade H2O. Efficiency of each reaction was evaluated by loading DNA in 0.8% agarose gel. Libraries were prepared with a two-step PCR procedure. For each 4C template, eight reactions of 100-200 ng were amplified by PCR using high fidelity Hot Start Phusion II DNA polymerase (ThermoFisher, F549) and long-template specific-bait primers containing the NexteraXT overhang sequences (Pparγ2_EcorI: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGATTAGTTTTATCTCTATTTAACTTGA Pparγ2_Csp6I: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATAGACTTGTTGAATAAATCACCT Dbp_HindIII: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAACCTGTTCTGTGGTGGATAAGC Dbp_Csp6I: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACTGGTTTTTGCCTGGAGGTC Pparα_HindIII: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCTCCAGGGTCTCAGTTTTGCA Pparα_Csp6I: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCATTTTCCTACCCTGGCCTGTA Srebp1c_HindIII: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCTGTCCCTGTCCCTTGCTGG Srebp1c_Csp6I: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGTCGGGGTAGCGTCTG), 2 min at 98°C, (15s at 98°C, 1 min at 48°C , 3 min at 72°C) x 30 cycles and a final extension of 7 min at 72°C. PCRs were pooled and for a second round of PCR using NexteraXT index kit. PCR products were size selected with 2% agarose gel to 200 bp-2kb, purified with gel purification kit (Qiagen) and fragment size distribution analyzed with bioanalyzer tapestation (Agilent).